Suppressor mutants demonstrate the metabolic plasticity of unsaturated fatty acid synthesis in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa PAO1 has two aerobic pathways for synthesis of unsaturated fatty acids (UFAs), DesA and DesB plus the oxygen independent FabAB pathway. The DesA desaturase acts on saturated acyl chains of membrane phospholipid bilayers whereas the substrates of the DesB desaturase are thought to be long chain saturated acyl-CoA thioesters derived from exogeneous saturated fatty acids that are required to support DesB-dependent growth. Under suitable aerobic conditions either of these membrane-bound desaturates can support growth of P. aeruginosa ∆fabA strains lacking the oxygen independent FabAB pathway. We previously studied function of the desA desaturase of P. putida in a P. aeruginosa ∆fabA ∆desA strain that required supplementation with a UFA for growth and noted bypass suppression of the P. aeruginosa ∆fabA ∆desA strain that restored UFA synthesis. We report three genes encoding lipid metabolism proteins that give rise to suppressor strains that bypass loss of the DesA and oxygen independent FabAB pathways.


INTRODUCTION
Biological membranes maintain appropriate fluidity to support membrane structure and function under various growth conditions.When adapting to an environment that requires greater membrane lipid rigidity, cells produce saturated fatty acids (SFA).In contrast, when membrane lipid function requires increased fluidity, more unsaturated fatty acids (UFAs) and/or branched-chain fatty acids are produced.
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen having membrane phospholipids composed of straight-chain SFAs and UFAs.In addition to the FabA-FabB oxygen-independent pathway, it has two oxygen-dependent desaturase pathways for UFA synthesis, DesA and DesB [1].DesA introduces a double bond in the Δ9 position of 16 : 0 FA chains esterified to the sn-2 position of existing phospholipids.In contrast, DesB desaturates exogenously supplied saturated acyl chains probably following conversion to 16 : 0-CoA and 18 : 0-CoA to produce 16 : 1Δ9-CoA and 18 : 1Δ9-CoA, respectively [1].The resulting unsaturated acyl-CoA are incorporated into phospholipids [2,3].Transcription of the desCB operon is regulated by the DesT repressor and acyl-CoA ligands.DesT binds the fabAB and desCB promoters [2,3].Saturated acyl-CoAs decrease the binding affinity of DesT to the desCB promoter thereby increasing desaturase levels and UFA production.In contrast unsaturated acyl-CoAs enhance DesT binding to the fabAB and desCB promoters to decrease UFA synthesis [4].However, the levels of transcription repression differ, <3-fold and ~9-fold for fabAB and desCB, respectively [1,2].
FA synthesis in both P. aeruginosa and E. coli results in acyl-ACP acyl chains of 16 or 18 carbons in length that are transferred to glycerol-3-phosphate to form phosphatidic acid, the key phospholipid precursor.The chains are transferred from acyl-ACP or acyl-CoA to the sn-1-position of glycerol-3-phosphate to form lysophosphatidic acid, LPA (formally 1-acyl-sn-glycerol-3-phos phate).In E. coli LPA is acylated in the sn-2-position to form phosphatidic acid by the essential PlsC LPA acyltransferase [5,6].However, many bacteria including pseudomonads encode multiple LPA acyltransferases that have only low amino acid sequence identity to PlsC, although active site and substrate binding residues are generally conserved.
Although we have a good understanding of the E. coli phosphatidic acid synthesis pathway, the Pseudomonas pathways remain largely unknown.As pointed out by Meredith and coworkers [7], 'fatty acid metabolism is especially complex in pseudomonads, with an unusually large genetic allocation to both the catabolism and anabolism of fatty acids.' These genomes encode multiple putative LPA acyltransferases, a few of which have been demonstrated to have LPA acyltransferase activity.However, unlike E. coli PlsC, no single LPA acyltransferase has been shown to be essential for growth.
A P. aeruginosa PAO1 strain lacking both the oxygen-independent pathway (∆fabA) and the DesA desaturase (∆desA) is a UFA auxotroph [1].However, when plated without UFA supplementation, the ∆fabA ∆desA strain suppressor colonies arose which restored UFA synthesis as well as growth in the absence of oleic acid.Sequencing of the genomes of four such suppressor strains showed mutations in three different genes.

Materials
DNA polymerase, restriction endonuclease, and T4 ligase were purchased from New England Biolabs.Sodium [1-14 C]acetate (specific activity, 57.0 mCi mmol −1 ) and [1-14 C]stearic acid (specific activity, 53.0 mCi mmol −1 ) were provided by Moravek, Inc.All the other reagents were of the highest available quality.Oligonucleotide primers were synthesized by Integrated DNA Technologies.

Materials
DNA polymerase, restriction endonuclease, and T4 ligase were purchased from New England Biolabs.Sodium [1-14 C]acetate (specific activity, 57.0 mCi mmol −1 ) and [1-14 C]stearic acid (specific activity, 53.0 mCi mmol −1 ) were provided by Moravek, Inc.All the other reagents were of the highest available quality.Oligonucleotide primers were synthesized by Integrated DNA Technologies.

Bacterial strains, plasmids, and growth conditions
The strains and plasmids used are given in Table 1.P. aeruginosa strains were grown at 37 °C and P. putida strains were grown at 30 °C in Luria-Bertani (LB) medium containing (in g l −1 ) (tryptone, 10; yeast extract, 5; NaCl, 10; pH 7.0) When required, antibiotics and inducers were added as follows (in μg ml −1 ): gentamicin, 90; isopropyl-β-d-thiogalactoside (IPTG), 2 40.Xylose was added at 50 mM.Oleate was used at a final concentration of 0.5 mM.Growth was assayed by colony formation on solid media.

Construction of complementation strains
The targeted gene fragment was amplified by PCR and the primers are given in Table 1, PadesB and PadesCB were digested with BamHI and HindII and ligated into the pMQ652 vector [8] digested with the same enzymes whereas lptA and its deletion derivatives, acdA and desT were digested with NdeI and HindIII, and ligated into vector pSRKGm [9] digested with the same enzyme.After screening and verification, the plasmids were transferred to the host cell by electroporation.For Pseudomonas, electroporation was performed as follows: a 5 ml culture of the host strain was grown overnight, 1 ml of the host strain culture was washed twice with 500 mM sucrose, concentrated to 100 µl, the complementation plasmid was added.The solution was transferred to the electroporation cuvette and put on ice for 10 min, after a 2.5 KV shock, 1 ml of fresh medium was added, the cultures were grown for 1 h at 37 °C (P.aeruginosa) or 30 °C (P.putida) and plated on media containing the appropriate selective antibiotic.

DNA sequencing
Genomic sequencing (Oxford Nanopore confirmed by Illumina sequencing) was done by the DNA Services Laboratory of the Carver Biotechnology Centre on this campus.Genomic DNAs was purified using the Promega DNA Purification Kit.Briefly, 1.5 ml of overnight culture strains were harvested and the bacterial genomic DNAs were purified according to the kit.Finally, DNA concentrations were determined by absorbance and DNA quality was checked by agarose gel electrophoresis.

Thin Layer Chromatography (TLC) analysis of phospholipid acyl chains
P. aeruginosa and P. putida strains were grown to OD 600 0.5 with 0.5 mM oleic acid, the cells were harvested by centrifugation, washed three times and incubated for another 3 h at 37 °C (P.aeruginosa) in the presence of [1-14 C]acetate (final concentration of 1 µCi ml −1 ) or [1-14 C]stearic acid (final concentration of 0.1 µCi ml −1 ).Cultures were lysed with methanolchloroform (2 : 1).The phospholipids were extracted with chloroform and dried under nitrogen.The fatty acyl groups on phospholipids were then converted to their methyl esters by transesterification with 25 % sodium methoxide, extracted into petroleum ether, taken to dryness under nitrogen, resuspended in hexanes and loaded onto 20 % silver nitrate TLC plates (Analtech) which

Growth of the suppressor strains
The suppressor strains required aerobic conditions for growth indicating that bypass of the ∆fabA ∆desA mutations is due to desaturation (Fig. 1a).Since P. aeruginosa has only two desaturases DesA and DesB [1] and DesA was eliminated by the ∆desA deletion, aerobic growth of the suppressor strains must involve DesB.Indeed, labelling of cultures with [ 14 C]acetate (Fig. 1b) or [ 14 C]stearate (Fig. 1c and data below) showed that all four suppressor strains synthesize ∆9 UFA species which are the products of DesB, the sole desaturase of the ∆fabA ∆desA strain.We used [ 14 C]stearate rather than [ 14 C]palmitate to directly assay DesB activity because desaturation of stearate gives the ∆9 C18 species which can readily be distinguished from the ∆11 C18 species formed by de novo synthesis whereas both palmitate desaturation and de novo synthesis produce ∆9 C16.

Genome sequences of the suppressor strains and the effects of their mutations on growth
The four suppressor strains were submitted for whole genome sequencing (Oxford Nanopore confirmed by Illumina sequencing).All suppressor strains retained the ∆fabA and ∆desA deletion alleles (Fig. 2).Strikingly, all four suppressor strains contained mutations in lptA (PA0005) which encodes an LPA acyltransferase that complements growth of the E. coli plsC(Ts) strain at the nonpermissive temperature [10].In three of the four suppressor strains (S1, S3 and S6) the lptA mutation is a premature stop codon that encodes a protein truncated by loss of 33 C-terminal residues whereas suppressor S2 has a different lptA mutation.The suppressor S2 LptA protein lacks the first 23 residues which would remove most of the hydrophobic helix one required for membrane binding and activity as shown in the Thermatoga maritima LPA [11].Moreover, suppressor S2 has a mutation in desT, the transcriptional repressor of desB .Two suppressor strains, S1 and S6, also contain a mutation in an acyl-CoA dehydrogenase (PA0506) that we have named AcdA.We believe that AcdA is a bona fide acyl-CoA dehydrogenase because it is 91 % identical to P. putida KT2440 dehydrogenase (PP_0368) where activity of the purified protein has been demonstrated [12].

Effects of the suppressor mutations and complementation by wild-type alleles
The mutations of the suppressor strains could be loss of function or gain of function alleles (albeit much less likely for deletions).This was tested by expression of the wild-type of each mutant allele from a tightly regulated promoter.Expression of the wildtype gene in each of the suppressor strains decreased or blocked both growth and UFA synthesis indicating that the suppressor mutations are loss of function.Expression of the wild-type DesT in suppressor two decreased growth and UFA synthesis even in the absence of induction indicating that the DesT of suppressor two is inactive (Fig. 3a).Similar results were seen upon expression of the wild-type AcdA (Fig. 3a).The most dramatic effect was that seen upon introduction of a plasmid encoding wild-type LptA into suppressor two.Induction of wild-type LptA expression resulted in complete blockage of growth and UFA synthesis (Fig. 3b) whereas the lptA deletion alleles (Fig. 2) did not block growth (Fig. 3c).These data also showed that the plasmids that expressed the wild-type lptA allele almost eliminated suppressor accumulation suggesting that routes to ∆fabA ∆desA suppression that do not involve LptA inactivation are rare.
The effects of the wild-type alleles on UFA synthesis were assayed by labelling of cultures with either [ 14 C]acetate or [ 14 C]stearate (Figs 4 and 5).Assay of de novo synthesis by [ 14 C]acetate labelling showed decreased levels of UFA synthesis when wild-type copies of desT or acdA were introduced into suppressor two and the levels of UFA synthesis decreased with induction (Fig. 4a).
As expected from the effects of expression of wild-type LptA on growth of suppressor two, induction of wild-type LptA expression abolished UFA synthesis (Fig. 5a).However, expression of either of the two lptA deletion alleles had no effect on UFA synthesis indicating that both are loss of function mutations (Fig. 5b).Direct assay of DesB desaturation by [ 14 C]stearate labelling (Figs 4b and 5b) showed more modest effects, although induction of expression of the DesT wild-type allele decreased UFA formation as expected from increased repressor levels.

DesB requires DesC for activity
Although we have interpreted desaturation in terms of DesB, DesB may not act alone.The desB gene is cotranscribed with desC, the product of which has been assigned an electron transfer-oxidoreductase role in the DesB desaturation reaction [1].However, the proposed DesC requirement for DesB function has not been tested.The P. putida F1 ∆fabA ∆desA strain provided a test because P. putida F1 lacks desCB [13].DesB was expressed alone or together with DesC in the absence of the oleic acid required by the ∆fabA ∆desA strain.Labelling with either [ 14 C]acetate or [ 14 C]stearate detected UFA synthesis only when both DesB and DesC were expressed, DesB alone was inactive (Fig. 6).These data indicate that P. putida F1 lacks a protein able to replace DesC.We tested the possibility that DesC might provide electron transfer-oxidoreductase function for P. putida F1 DesA but this was not the case.The P. putida F1 DesA expressing DesC remained unable to support growth of a ∆fabA strain (data not shown).

DISCUSSION
All four suppressor strains have a loss of function mutation in lptA.Expression of P. aeruginosa PAO1 lptA is known to complement growth of the E. coli plsC(Ts) strain demonstrating that it is a functional LPA acyltransferase [10].The P. aeruginosa PAO1 lptA deletion strain grew somewhat more slowly than the parent at 37 °C but much more poorly than the wild-type parent at 11 °C [10].fatty acid composition of the ∆lptA strain differed somewhat from that of the wild-type strain in distribution between C16 and C18 chains.Complementation of the ∆lptA strain with a plasmid expressing wild-type LptA restored the wild-type composition [10].In P. fluorescens two LPA acyltransferase genes called hdtS and patB were also found to complement growth of the E. coli plsC(Ts) strain [14].HdtS is 70 % identical to P. aeruginosa PAO1 LptA whereas there is no convincing P. aeruginosa PAO1 PatB homolog.A ∆hdtS strain showed a modest growth defect at 30 °C that was corrected by expression of E. coli PlsC whereas the ∆patB strain grew at 30 °C but poorly at 38 °C [14].Note that P. aeruginosa PAO1 and P. fluorescens each have several annotated LPA acyltransferase genes in addition to those studied: five and six, respectively (www.microbesonline.org).Currently no single Pseudomonas LPA acyltransferase has been shown to be essential growth.(c) The deleted lptA alleles, unlike the wild-type gene, fail to block growth of suppressor strain two.Up and Dn refer to the upper and lower lptA mutants as depicted in Fig. 2.These data indicate that all mutant alleles of suppressor strain two encode proteins that lack function.
A complex situation exists for the acdA of suppressor strains S1 and S6 because P. aeruginosa PAO1 encodes at least ten, perhaps as many as twenty acyl-CoA dehydrogenases.This multiplicity is consistent with the ability of P. aeruginosa PAO1 to grow on a large variety of aromatic and aliphatic compounds as carbon and energy sources.The rings of the aromatic compounds are cleaved to give aliphatic compounds which together with the aliphatic compounds are converted to acids and Expression was driven by the lac promoter of vector pSRKGm [9].degraded like pathways [15].However, the Pseudomonas acyl-CoA dehydrogenases have overlapping specificities which complicates assigning distinct metabolic roles.This is illustrated by a study in P. putida KT2440 which is thought to encode twenty-one acyl-CoA dehydrogenases [16].Deletion derivatives of four of these genes were constructed and growth of the resulting strains on a range of alkanoic acids from propionic (C3) to palmitic (C16) acids was measured.All four deletion strains grew on all sixteen acids.Only one deletion strain had a phenotype and that was a modestly decreased ability to utilize C3-C9 acids [16].
The only mutation of a suppressor strain that lacks putative homologs is the desT mutation of suppressor two which encodes a defective protein.DesT is a repressor that regulates the DesB and FabAB pathways [2].(Only DesB is regulated in these strains due to the fabA deletion.)The desT mutation of suppressor two removed the desT termination codon resulting in a 66-residue extension of the protein.AlphaFold [17] predicts that the extension is essentially unstructured although some models contain a short (three turn) helix.The most likely consequence of the extension is inhibition of the DesT dimerization required for tight promoter binding [1,4].This would result in derepression of desB and an increased level of desaturase.As expected, overproduction of wild-type DesT repressed desaturation in suppressor two as measured by [ 14 C]stearate labelling (Fig. 4b).
A model (Fig. 7) based on these data that could explain suppression of the ∆fabA ∆desA mutations is that the lptA mutations result in accumulation of C16 and C18 saturated acyl-ACPs and these are desaturated either directly or indirectly by DesB before incorporation into phospholipid.Soluble plant desaturases act on stearoyl-ACP and thus acyl-ACPs are plausible DesB substrates [18].Another possibility is that the saturated acyl-ACPs could be transacylated to acyl-CoAs, a reaction catalysed by 3-ketoacyl-ACP synthases [19,20] and are subsequently desaturated.(Although reasonable, there is no direct evidence that acyl-CoAs are DesB substrates.)In the model the role of the other mutations present in the suppressor strains is to optimize the effects of the LptA mutations.A secondary effect is consistent with the weaker effects of complementation with the wild-type alleles of acdA and desT mutations on UFA synthesis.The acdA acyl-CoA dehydrogenase mutations would prevent degradation of the accumulated acyl-ACPs either directly or after their conversion to acyl-CoAs.The desT lesion of suppressor two would result in an increase in DesB desaturase levels.
The multiple annotated LPA acyltransferase and acyl-CoA dehydrogenase genes of P. aeruginosa PAO1 are a puzzle.What is special about LptA and AcdA?Why were these genes inactivated rather than genes encoding putative orthologs?The P. aeruginosa PAO1 ∆lptA strain has a growth deficiency and an altered fatty acid composition (increased C18 chains at the expense of C16 chains) [10] whereas the only other LPA acyltransferase deletion strain, ∆patB of P. fluorescens, does not [14].
The slow growth reported for the P. aeruginosa PAO1 ∆lptA strain argues that strains lacking LptA may accumulate acyl-ACPs because LPA acyltransferase activity is limiting despite the other putative P. aeruginosa PAO1 LPA acyltransferases.There seems no straightforward explanation for multiple LPA acyltransferases, although at least one other acyltransferase must be active in the P. aeruginosa PAO1 ∆lptA strain since LptA is not essential [10].The phospholipid acyl chain composition of P. aeruginosa PAO1 is essentially the same as that of E. coli which has only a single LPA acyltransferase, PlsC.Hence, it seems unlikely that pseudomonads would need an array of acyltransferases for attachment of specific acyl chains.The P. putida acyl-CoA dehydrogenase (PP_0368) that is 91 % identical to P. aeruginosa PAO1 AcdA is significantly more active on palmitoyl-CoA and stearoyl-CoA than on any of fourteen other substrates tested [12].Hence, the substrate specificity of AcdA may have provided a selection for its inactivation.
Another puzzle is that unlike P. aeruginosa, desB is an essential gene in Acinetobacter baumannii, a related obligately aerobic bacterium.A. baumannii is an unusual bacterium that lacks a de novo UFA synthesis pathway and must obtain UFA by desaturation.The genome is annotated as encoding eight desaturases, one of which, DesB, is essential for more than trace growth in the absence of UFA supplementation [21].The A. baumannii desB is found in the same genome context as in P. aeruginosa and is regulated by a DesT homologue.A. baumannii also encodes a DesA desaturase which is not essential [21].These phenotypes are the opposite of those in P. aeruginosa where in the absence of UFA synthesis (a ∆fabA strain) desA is essential whereas desB is not [22].The A. baumannii DesA seems weaker (or more poorly expressed) than P. aeruginosa DesA because a ∆desB strain has less than one-third the UFA content of the parental strain and grows very poorly in the absence of UFA supplementation [21].DesA is responsible for this residual UFA production because a ∆desA ∆desB strain perishes in the absence of UFA supplementation [21].Both P. aeruginosa and A. baumannii are Pseudomonadales isolated from human wounds and thus the switching of the physiological roles of the DesA and DesB in the two bacterial species is difficult to rationalize.

Fig. 1 .
Fig.1.Suppressors of the P. aeruginosa PAO1 ∆fabA ∆desA strain.(a) Suppressor colonies arose on LB medium without oleic acid and grew well upon restreaking on the same medium but only under aerobic conditions.Labelling of suppressor strain one cultures with [1-14 C]acetate (b) or [1-14 C]stearic acid (c) showed UFA synthesis was restored in the suppressor strain.The other three suppressors showed similar growth and in the absence of oleic acid.UFA synthesis was also restored in the other three suppressors as seen for suppressor S2 in Fig.4.

Fig. 2 .
Fig.2.The mutations present in the suppressor strains.All four strains have a mutation in lptA (PA0005).Deletion mutations were found in acdA (PA0506) encoding an acyl-CoA dehydrogenase in suppressor strains S1, S3 and S6 whereas suppressor S2 also contained a mutation in desT (PA4890) the repressor of desB expression.Our work focused on suppressor two because it contains the two best-defined mutations in the lptA and desT genes of those altered in the suppressor strains.

Fig. 3 .
Fig. 3. Inhibition of the growth of suppressor two (Supp) by expression of the wild-type copies of the mutant genes.(a) Expression of either the AcdA acyl-CoA dehydrogenase or the DesT repressor inhibited growth of the suppressor strain two upon induction of expression of the wild-type gene.(b) Growth of suppressor strain two was completely blocked upon induction of wild-type lptA.Addition of oleic acid only very weakly supported growth.(c)The deleted lptA alleles, unlike the wild-type gene, fail to block growth of suppressor strain two.Up and Dn refer to the upper and lower lptA mutants as depicted in Fig.2.These data indicate that all mutant alleles of suppressor strain two encode proteins that lack function.

Fig. 4 .
Fig. 4. Effects of expression of the wild-type copies of acdA or desT on UFA synthesis.(a) Labelling with [ 14 C]acetate.(b) Labelling with [ 14 C]stearate.Expression was driven by the lac promoter of vector pSRKGm[9].

Fig. 5 .
Fig. 5. Effects of expression of the wild-type copy of lptA or deletion derivatives of lptA on UFA synthesis.(a) Labelling with [ 14 C]acetate.(b) Labelling with [ 14 C]stearate.Expression was driven by the lac promoter of vector pSRKGm [9].

Fig. 6 .
Fig. 6.Desaturation by DesB requires DesC.The P. putida ∆fabA ∆desA strain [13] was transformed with plasmids encoding either desB or desCB.The left panel is [ 14 C]acetate labelling whereas the right panel is [ 14 C]stearate labelling.The genes were expressed from the xylose inducible promoter of vector pMQ652 [8] in the presence of 50 mM xylose.

Table 1 .
Strains, plasmids and oligonucleotide primers r , Shuttle vector, pBBR1, with xutR-Pxut and multicloning site.[8] pHJ85 P. aeruginosa desB expression plasmid derived from pMQ652 This work pHJ177 P. aeruginosa desCB expression plasmid derived from pMQ652 This work pHJ312 P. aeruginosa acdA expression plasmid derived from pSRKGm This work pHJ12 P. aeruginosa lptA expression plasmid derived from pSRK Gm r This work pHJ100 P. aeruginosa lptA (224 aa up deletion) expression plasmid derived from pSRK Gm r This work pHJ101 P. aeruginosa lptA (224 aa down deletion) expression plasmid derived from pSRK Gm r